The present invention relates to a process for the production of chimeric antibodies using recombinant DNA techniques.
In the present application, the term chimeric antibody is used to describe a protein comprising at least the antigen binding portion of an immunoglobulin molecule (Ig) attached by peptile linkage to at least part of another protein.
In recent years, advances in molecular biology based on recombinant DNA techniques have provided processes for the production of a wide range of heterologous polypeptides by transformation of host cells with heterologous DNA sequences vhich code for the production of the desired products.
EP-A-O 088 994 (Schering Corporation) proposed the construction of recombinant DNA vectors comprising a ds DNA sequence vhich codes for a variable region of a light or a heavy chain of an Ig specific for a predetermined ligand. The ds DNA sequence is provided with initiation and termination codons at its 5xe2x80x2- and 3xe2x80x2-termini respectively, but lacks any nucleotides coding for amino acids superfluous to the variable region. The ds DNA sequence is used to transform bacterial cells. The application does not contemplate the production of chimeric antibodies.
EP-A-1 102 634 (Takeda Chemical Industries Limited) describes the cloning and expression in bacterial host organisms of genes coding for the whole or a part of human IqE heavy chain polypeptide, but does not contemplate the production of chimeric antibodies.
EP-A-0 125 023 (Genentech Inc. et al.), which was published after the priority date of the present application, proposes the use of recombinant DNA techniques in bacterial cells to produce Ig""s which are analogous to those normally found in vertebrate systems and to take advantage of the gene modification techniques proposed therein to construct chimeric Ig""s or other modified form of Ig.
It is believed that the proposals set out in the above Genentech application did not lead to the expression of any significant quantities of Ig polypeptide chains, nor to thes production of Ig activity, nor to the secretion and assembly of the chains into the desired chiaeric Ig""s.
The production of monoclonal antibodies was first disclosed by Kohler and Milstein (Kohler, G. and Milstein, C., Nature, 256, 495-497, 1975). Such monoclonal antibodies have found widespread used not only as diagnostic reagents (see, for example, xe2x80x98Immunology for the 80s, Eds. Voller, A., Bartlett, A., and Bidwell, D., MTP Press, Lancaster, 1981) but also in therapy (see, for example, Ritz, J. and Schlossman, S. F., Blood, 59, 1-11, 1982).
The recent emergence of techniques allowing the stable introduction of Ig gene DNA into myeloma cells (see, for example, Oi, V. T., Morrison, S. L., Herzenberg, L. A. and Berg, P., PNAS USA, 80, 825-829, 1983; Neuberger, M. S., EMBO J., 2, 1373-1378, 1983; and Ochi, T., Hawley, R. G., Hawley, T., Schulman, M. J., Traunecker, A., Kohler, G. and Hozumi, N., PNAS USA, 80, 6351-6355, 1983), has opened up the possibility of using in vitro mutagenesis and DNA transfection to construct recombinant Ig""s possessing novel properties.
However, it is known that the function of an Ig molecule is dependent on its three dimensional structure, which in turn is dependent on its primary amino acid sequence. Thus, changing the amino acid sequence of an Ig may adversely affect its activity. Moreover, a change in the DNA sequence coding for the Ig may affect the ability of the cell containing the DNA sequence to express, secrete or assemble the Ig.
It is therefore, not at all clear that it will be possible to produce functional altered antibodies by recombinant DNA techniques.
Simiar considerations apply to other proteins. It therefore cannot be expected that fusion of a gene coding for at least part of an Ig with a gene coding for at least part of another protein will lead expression of any protein, let alone expression of protein which can be secreted and assembled to give a functional chineric antibody.
However, the present inventors have now discovered unexpectedly that it is possible to produceby recombinant DNA techniques secreted, assembled chimeric antibodies in which both parts of the protein are functional.
This surprising result is achieved by the use of the process of the present invention, which comprises:
a) preparing a replicable expression vector including a suitable promoter operably linked to a DNA sequence comprising a first part which encodes at least the variable region of the heavy or light chain of an Ig molecule and a second part which encodes at least part of a second protein;
b) if necessary, preparing a replicable expression vector including a suitable promoter operably linked to a DNA sequence which encodes at least the variable region of a complementary light or heavy chain respectively of an Ig molecule;
c) transforming an imortalised mammalian cell line with the or both prepared vectors; and
d) culturing said transformed cell line to produce a chimeric antibody.
The immortalised cell line is preferably of lymphold origin, such as a myeloma, hybridoma, trioma or quadroma cell line. The cell line may also comprise a normal lymphoid cell, such as a B-cell, which has been inmortalised by transformation with a virus, such as the Epstein-Barr virus. Most preferably, the immortalised cell line is a myeloma cell line or a derivative thereof.
It is known that some immortallsed lymphoid cell lines, such as ayeloma cell lines, in their normal state secrete isolated Ig light or heavy chains. If such a cell line is transformed with the vector prepared in step a) of the process of the invention, it will not be necessary to carry out step b) of the process, provided that the normally secreted chain is complementary to the part of the Ig molecule encoded by the first part of the vector prepared in step a).
However, where the immortalised cell line does not secrete or does not secrete a complementary chain, it will be necessary to carry out step b). This step may be carried out by further manipulating the vector produced in step a) so that this vector encodes not only the fusion of variable region and second protein, but also the complementary variable region. However, preferably step b) is carried out by preparing a second vector which is used to transform the immortalised cell line.
The techniques by which such vectors can be produced and used to transform the immortalised cell lines are well known in the art, and do not form any part of the invention. However, they are well illustrated in the following Examples.
In the case where the immortalised cell line secretes a complementary light or heavy chain, the transformed cell line may be produced by transforming a suitable bacterial cell with the vector and then fusing the bacterial cell with the immortalised cell line, e.g. by Spheroplast fusion.
The first part of the DNA sequence say be joined directly to the second part thereof. Alternatively, the first part may be joined to the second part by an intervening sequence which encodes a specific cleavage sequence, for instance a Factor Xa cleavage sequence as described in our copending European patent application No. 85303414.8. Reference may be made to this application for further discussion of the use to specific cleavage sequences.
The second part of the DNA sequence may encode:
i) at least part, for instance the constant region of a heavy chain, of an Ig molecule of different species, class or subclass;
ii) at least the active portion or all of an enzyme;
iii) a protein having a known binding specificity;
iv) a protein expressed by a known gene but whose sequence, function or antigenicity is not known; or
v) a protein toxin, such as ricin.
The chimeric antibody produced in case i) above will be of use in a number of applications. For instance, an established cell line may produce an Ig molecule having a useful specificity. However it may be of a class which is diagnostically or therapeutically undesirable, or it may not be secreted in useful quantities. For instance, Ig of glass M is known to be difficult to use in rapid immunoassay techniques and is generally inconvenient for use in therapy, whereas Ig of class G can be readily used in these techniques. Therefore, it would be possible to produce a useful immunoassay reagent or therapeutic agent by replacing the IgM heavy chain constant region with an IgG heavy chain constant reglon. A particular example of such use would be in the production of an chimeric antibody having an anti Rh specificity, derived from an IgM secreting myeloma and IgG reactivity provided by an IgG derived heavy chain constant region.
Alternatively, the chimeric antibody could comprise an IgG derived variable region and an Ig E derived heavy chain constant region. Such a chimeric antibody could be used to investigate the action of IgE on mast cells, in diagnostic assays, for instance calibrating test procedures or, in therapy, to inhibit allergic reactions caused by the action of normal IgE molecules on mast cells.
In another alternative, the chimeric antibody may be used toalter the complement binding activity of an antibody, again by changing the heavy chain constant region.
In a further alternative, the chimeric antibody may be constructed to resemble an (Fabxe2x80x2)2 fragment of a normal antibody.
The chimeric antibody produced in case ii) above may be used, in particular, in an enzyme linked immunoassay (ELISA) system, in place of the present use of separate antibodies and enzymes.
The chimeric antibody produced in case iii) above may also be used in immunoassays. For instance, the protein may have a binding specificity for an easily detectable label, such as a heavy or radioactive metal or a dyed or dyeable molecule. For instance, it would be possible to produce a divalent chimeric antibody having a different variable region at each end, the ends being connected by at least a part of a constant region.
The chimeric antibody produced in case iv) above may be used to investigate the products of known genes. For instance, it may be known that a particular gene produces a protein involved in the surface marking of a cell. However, the exact nature of the surface marker may not be known. The chimeric antibody produced in case iv) will comprise the protein product of the gene, which can therefore be more readily characterised. Moreover, antibodies to this gene product could be raised and used to investigate with certainty the distribution of the gene product on a cell surface.
The chimeric antibody produced in case v) above will clearly be of use in therapy, for instance as a targetted cytotoxic agent for cancer therapy. In this respect reference may be made to an article by Thorpe et al. (Thorpe, P. E., Edwards, D. C., Davies; A. J. S. and Ross, W. C. J., in xe2x80x98Monoclonal Antibodies in Clinical Medicinexe2x80x99, 167-201, Eds., McMichaele A. J. and Fabre, J. W., Academic Press, 1982).
The chimeric antibodies produced by the present process, especially where the DNA sequence encodes a specific cleavage site, may be used for purifying the second protein. For instance, the variable region may be made specific for a hapten whicn can be immobilised on a chromatography medium. The chimeric antibody can then be immobilised by affinity chromatography and contaminating material can be washed away. The second protein can then be cleaved from the variable region either before or after the variable region is eluted from the chromatography medium.
The chimeric proteins of the type referred to in cases ii) to iv) above and in the preceding paragraph also comprise aspects of the present invention.